Push column chromatography apparatus

ABSTRACT

An apparatus for chromatography of DNA, RNA, proteins and other molecules includes the use of a column adapted to hold a chromatography material and a sample to be filtered. A pneumatic pressure differential is applied across the column and the sample is urged through the chromatography material. A selected portion of the sample may then be collected.

This application is a divisional of application Ser. No. 08/084,533,filed Jun. 28, 1993, now U.S. Pat. No. 5,378,360, which is a divisionalof application Ser. No. 07/827,995, filed Jan. 30, 1992, now U.S. Pat.No. 5,378,359, which is a continuation of application Ser. No.07/292,808, filed Jan. 3, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an apparatus and methodology for thechromatography of materials, and in particular, chromatography based onmolecular size, affinity and the like as used, for example, in thepurification, separation or isolation of DNA and RNA fragments, proteinsand other molecules.

2. Background Art

Removing unincorporated nucleotides from DNA and RNA fragments,isolating RNA fractions, purifying proteins and other macromolecules,are important procedures having a variety of applications. In DNA andRNA synthesis, unincorporated nucleotides must often be removed whenconstructing nick-translated probes, RNA probes and end-labeledoligonucleotides, as well as "filled-in" DNA fragments. It is importantto separate the unincorporated free-nucleotides from the labeled probeas unincorporated label may bind to the solid support, resulting inunacceptably high levels of background noise. Isolation of RNA fractionsmay be employed in the separation of, for example, polyadenylated RNAfrom nonpolyadenylated RNAs. The use of chromatography methods toisolate and identify proteins and other macromolecules is another wellknown application.

Current chromatography methods, used particularly in connection with DNAand RNA synthesis, include ion-exchange chromatography severalvariations of eel chromatography and others. Each has its owndisadvantages. For example, ion-exchange methods require a number ofsteps which may result in a significant investment of time and, in thecase of radio-labeled nucleotide filtering, extensive handling ofradioactive material. Conventional gel-chromatography "drip" columns aretedious, requiring time to both pour and run. Spin columns, a variationof the "drip" column, are somewhat faster, but risk radiation exposureand contamination in the case of radionucleotide chromatography, and mayyield less reliable results.

An alternative chromatography approach which avoids the aforementioneddifficulties would therefore be desirable.

SUMMARY OF THE INVENTION

The present invention is directed to an apparatus and method forpurifying, isolating and separating materials using gel chromatography.To that end, a chromatography material and a sample may be loaded into acolumn and pneumatic pressure applied to urge the sample through thechromatography material, whereby portions of the sample may be collectedby the chromatography material and other portions excluded. In oneembodiment, a positive pneumatic pressure is provided and in a secondembodiment a negative pressure is applied. Additionally, a novel supportstructure may be employed to support the column during chromatography.The sample may thus be quickly and reliably treated.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exploded perspective view of an apparatus constructed inaccordance with the present invention comprising a column, pressureinducing means, a collection vial and associated support structure.

FIG. 2 is a cross-sectional view of the apparatus of FIG. 1 in a loadedposition.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Referring to FIGS. 1 and 2, a chromatography apparatus constructed inaccordance with the present invention comprises a generally disk shapedbase 10 having a pair of retainers 12 and a generally cylindrical vialholding assembly 20 mounted thereon. Centrally located in the vialholding assembly 20 is a cylindrical chamber 22 for supporting acollection vial 30, into which the eluent from the column may becollected. The vial 30 may be a decapped Eppendorf tube or othersuitable collection means. Removably mounted to the base 10, andslideably engaging the exterior wall of the vial holding assembly 20, isa generally cylindrical column support assembly 40. The column supportassembly 40 includes a central aperture 42 formed in the generallyplanar upper surface 44 thereof. As shown in FIG. 2, the supportassembly may have a resilient collar 46, such as an "O" ring or thelike, positioned circumferentially adjacent the aperture 42, and acollar retainer 48 adapted to retain the collar 46 adjacent the aperture42. Alternatively, as shown in FIG. 1, the collar 46 and the retainer 48may be eliminated.

Optionally, a generally cylindrical pressure inducing means supportassembly 50 may be removably mounted on the base 10. The supportassembly may comprise a central aperture 52 formed in the generallyplanar upper surface 54 thereof, and is configured to slideably engagethe exterior wall of the column support assembly 40. The aperture 52 ispreferably axially aligned with the aperture 42 in the column supportstructure 40, which itself is preferably axially aligned with thechamber 22 in the vial holding assembly 20.

Alternatively, as shown in FIG. 1, the support assembly 50 may includean upper surface 54 having no aperture therein. The support assembly 50may be further provided with a pair of locking tabs 54 adapted to engagethe retainers 12 on the base 10 to lock the support assembly 50 in placeduring use. Other suitable locking mechanisms, such as threads, couldalso be employed. The assemblies 20, 40 and 50 may be formed of aradiation shielding material or, preferably, are constructed to fitsecurely inside a beta shield device. Molded plastic materials have beenfound suitable although other materials may also be employed.

Supported by the column holding assembly 40 above the vial 30 is asubstantially tubular chromatography column 60. The column 60 may beabout 1 ml in size, having a preferred internal diameter of about 5 mmand a preferred length of about 100 mm, and comprises openings 62 and64, respectively, at each end thereof. An annular lip 66 may be providedcircumferentially adjacent the upper opening 62, as shown in FIG. 1. Theupper opening 62 is adapted to receive a chromatography material 70 anda sample 80 to be filtered. The lower opening 64 has an area of reducedcross-section adapted to prevent passage of the chromatography material70 while permitting passage of the sample 80. Additionally, a screen orfilter 68, comprising, for example, glass wool, may be employed toretain the chromatography material 70 within the column 60. Preferably,for a tubular column, the internal diameter should not exceed about 10mm for most chromotography applications, lest the surface tension of thesample be insufficient to prevent effervescence and consequent loss ofpressure through the sample. Increasing the length of the column 60should enhance the degree of separation. Preferably, the column shouldbe no less than about 60 mm in length.

Pneumatic pressure inducing means 90, in this case a syringe, may beattached to the upper opening 62 of the column 60. As shown in FIG. 1,the syringe 90 may include a series of threads 92 which engage theannular lip 66 of the column 60 to retain the column 60 and the syringe90 in mutual engagement. In a first embodiment of the invention, thesyringe 90, having the plunger 94 withdrawn as shown in FIG. 2, may beattached to the column 60 and a positive pneumatic pressure differentialapplied between the openings 62 and 64 of the column 60 by depressingthe plunger. In that case, the support assembly 50 may be placed overthe support assembly 40. As the assembly 50 is lowered, its uppersurface 54 (if no aperture 52 is provided) will contact the plunger 94and automatically depress same until the bottom of the support assembly50 meets the base 10. At that point the support assembly 50 may betwisted until the locking tabs 54 mate with the retainers 12. If anaperture 52 is provided in the support assembly 50, the plunger 94 willextend therethrough and may be manually depressed. In an alternativeembodiment, not shown, the pressure inducing means 90 may be attached tothe lower opening 64 of the column (the column may be removed from thesupport assembly 40 in that case) and the sample drawn through thecolumn by a negative pressure differential between the openings 62 and64.

The chromatography procedure may be commenced by removing the columnholding assembly 40 from the base 10 and inserting the collection vial30 into the aperture 22 in the vial holding assembly 20. The columnholding assembly 40 is then returned to the base 10. The column 60 isinserted into the aperture 42 in the column holding assembly 40 andpositioned so that the lower end of the column extends into thecollection vial 30. An appropriate chromatography material may then beintroduced into the column 60 using the syringe 90, or other suitablemeans.

If unincorporated nucleotides are to be removed from DNA or RNAfragments, gel chromatography material such as a polysaccharide orpolyacrylamide, having a selected degree of internal porosity, may beemployed. The sample containing DNA or RNA fragments (large molecules)and unincorporated nucleotides (smaller molecules) may be introducedinto the top of the column 40 using a suitable pipetting device.Capillary action draws the sample into the upper portion of thechromatography material, i.e., between the "beads" comprising thematerial as shown in FIG. 2. Preferably, if a 1 ml column is employed,about 10-50 ul, preferably 50 ul, and no more than about 200 ul, ofsample may be introduced. With the column thus prepared, the syringe 90may be attached with the plunger fully extended to the column 20. Theplunger may then be firmly depressed (with or without use of the supportassembly 50) until the sample is pushed through the column into thecollection vial 30. If the support assembly 50 is employed, the supportassembly locking mechanism can be actuated to retain the plunger in afully depressed condition. As the sample proceeds through thechromatography material, the smaller molucules, for example,unincorporated nucleotides, are partitioned into the pores in thechromatography material while the large molucules, for example, DNA orRNA fragments, are excluded. The eluent from the column should besubstantially free of unincorporated nucleotides.

A similar procedure may be employed for affinity chromotagraphyapplications such as hybridization of complimentary strands of nucleicacids. For example, to separate polyadenylated RNA fromnonpolyadenylated RNA, oligo(dT)-cellulose may be employed as achromatography material. Under appropriate buffer conditions, thedesired polyadenylated RNA will bind with the oligo (dT)-cellulosechromatography material while the nonpolyadenylated RNAs will be elutedinto the collection vial 30. The polyadenylated RNA can be recovered bya second buffer condition. Other affinity chromotography applicationsinclude the purification of specific nucleic acid sequences, forexample, viral genomic sequences, by generating complementaryoligonucleotides.

Thus, an apparatus and chromatography method employing a pneumaticpressure differential have been disclosed. While embodiments andapplications of this invention have been shown and described, it wouldbe apparent to those skilled in the art that many more modifications arepossible without departing from the inventive concepts herein. Forexample, although purification of DNA and RNA fragments and separationof polyadenylated from nonpolyadenylated RNAs has been disclosed, manyother chromotography applications would be possible. The invention,therefore, is not to be restricted except in the spirit of the appendedclaims.

What is claimed is:
 1. An apparatus for supporting a chromatography column pressurized by a syringe and for supporting a collection vial at a discharge end of the column, the apparatus comprising:a base; a vial support coupled to the base and having a chamber therein adapted for receiving and supporting a collection vial; a column support including a first hollow cylinder with an open lower end that slidably couples to the vial support on the base and an upper end with a hole therein adapted to support the column; and a syringe support including a second hollow cylinder with an open lower end that slidably couples to the column support and an upper end adapted to engage the syringe.
 2. The apparatus as recited in claim 1 wherein the column has a flange at an upper end thereof and said column support engages the column flange to longitudinally support the column, and wherein said syringe support has an upper surface inside the cylinder that engages the top of a plunger of the syringe so that the syringe support displaces the plunger downward when the syringe support is displaced downward.
 3. The apparatus as recited in claim 2 further comprising means for releasably locking the syringe support to the base when the syringe support is displaced downward into contact with the base.
 4. The apparatus as recited in claim 1 wherein said hole in the upper end of the column support laterally supports the column, and wherein the syringe has a flange at an upper end thereof and said syringe support includes a hole in the upper end that engages the syringe flange to longitudinally support the syringe.
 5. The apparatus as recited in claim 1 wherein said column support and said syringe support are composed of radiation shielding materials. 